Sample: CBA.G4F-426-L3W

Analytes:

Overview

The gel image below shows the locations of the analyte chains presented in this report.

Alpha-1-antitrypsin (P01009)

From: Human plasma protein N-glycosylation

Alpha-1-antitrypsin (AAT), also known as alpha-1-protease inhibitor, alpha-1-antiproteinase or serpin A1, consists of 418 amino acids (including a 24 amino acid signal peptide) with an apparent mass of 51 kDa (including glycosylation). It is mainly produced in the liver by hepatocytes, but is also synthesized in monocytes, intestinal epithelial cells, and in the cornea [52, 208–211]. Due to its small size and polar properties, the glycoprotein can easily move into tissue fluids [52]. In healthy individuals, a plasma level of approximately 1.1 mg/mL is found, but the concentration can increase three- to four-fold during inflammation [212–215]. AAT occurs as three different amino acid sequences, of which the first is set as the standard sequence. Form 2 differs in the amino acid sequence 356–418 and form 3 lacks the amino acid sequence 307–418.

Haptoglobin (P00738)

From: Human plasma protein N-glycosylation

Haptoglobin (Hp) is a 406 amino acid (18 amino acid signal peptide) acute-phase glycoprotein with a peptide backbone of 45 kDa. It is synthesized in the liver by hepatocytes as a single polypeptide chain and is also found in skin [304, 305]. During its synthesis, Hp is cleaved into a light a chain and a heavy ß chain that are connected via disulfide bonds. Two variants of the a chain originating from the sequence Val19-Gln160 and differing by the subsequence Glu38-Pro96 can exist, a1 having this subsequence once while a2 has it twice, resulting in a chains of 83 or 142 amino acids with a respective molecular mass of 9 and 16 kDa. The 40 kDa ß chain is made of 245 amino acids originating from the sequence Ile162-Asn406 [306, 307]. The combination of different allelic variants of the a chain (a1 and a2) with ß chain(s) creates the polymorphism observed in Hp. There are three major Hp phenotypes called Hp1-1, Hp2-1 and Hp2-2. They have a configuration of (a1ß)2, (a1ß)2 + (a2ß)n = 0, 1, 2, … and (a2ß)n = 3, 4, 5, …, respectively, which are observed at different ratios among ethnicities [118, 308–310]. Caucasians have around 13 % of phenotype Hp1-1, 46 % of Hp2-1 and 41 % of Hp2-2. Hp is typically found at a plasma levels in the range of 0.6–2.3 mg/mL with a mean of 1.32 mg/mL [118]. Elevated Hp levels have been reported with inflammation and malignant diseases [308, 311, 312]. It should be taken into account that the concentration as well as the molecular mass including glycosylation may vary among phenotypes (86–900 kDa) [118]. The half-life of Hp is found to be on average four days.

The major function of Hp is to protect tissues from oxidative damage by capturing hemoglobin [307, 313]. It has been reported that Hp polymorphism has an effect on its physiological properties, for instance Hp1-1 binds hemoglobin stronger than Hp2-2 [314]. Certain diseases seem to be dependent on the polymorphism, as individuals with the Hp1-1 phenotype seem to have a higher concentration of induced antibodies in their plasma after vaccination, infections or liver diseases compared to the other phenotypes [118, 310].

Transferrin (P02787)

From: Human plasma protein N-glycosylation

Serotransferrin (STF), also known as transferrin, ß1 metal binding globulin or siderophilin, is a 698 amino acid protein (19 amino acids of which are signal peptide) with a molecular mass of approximately 77 kDa (without glycosylation) [8, 337]. The protein consists of two globular domains, the N-lobe and the C-lobe which divided into two subdomains each (N1, N2, C1 and C2). The two main domains are connected by a short linker peptide [337–339]. The N-lobe is 336 amino acids in size and spans from Val25 to Glu347, while the C-lobe is 343 amino acids long and ranges from Val361 to Lys683 [337]. The lobes can interact to form a hydrophilic metal ion binding site [337]. STF is mostly produced by hepatocytes, although other tissues have also shown expression, albeit at significantly lower amounts [337]. The plasma concentration is highly stable from the age of 2 years on, with a range between 2 and 3 mg/mL [337, 340]. Levels may increase during pregnancy up to 5 mg/mL [141].

Statistical Process Control

From: An introduction to statistical process control in research proteomics

Background: Statistical process control is a well-established and respected method which provides a general purpose, and consistent framework for monitoring and improving the quality of a process. It is routinely used in many industries where the quality of final products is critical and is often required in clinical diagnostic laboratories [1,2]. To date, the methodology has been little utilised in research proteomics. It has been shown to be capable of delivering quantitative QC procedures for qualitative clinical assays [3] making it an ideal methodology to apply to this area of biological research.

Control charts and possibly image areas from the standard.

Audit Logs:

 Run overview

Run #	208
Sample / Study	Human plasma (Lot: SLCC1673)/ NUBZ64100, NUCX182416, NUBZ648698, NUAQ387360, NUFH402412, NUFT865086, NUFR670624, NUFT766262, NUFU715690, NUFT770122, NUFT766280, NUCX210223
Completion (Scan) Date	25/09/2020
Comments	OOD GE strips, biotium dye, sample load 2uL. Pre-mix buffers. Standard IEF with cuploading. Standard 2D with type 2 water. 4% equilibration buffer.

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 Labelling

Technician:EA & NH

Sample: 2µL Human Plasma

Solution / reagent	Batch / Lot #	Reference
DIGE labelling buffer	6	LB-RS-06
Tris-HCl pH 8.5	2	LB-RS-02
DMF	STBH7014
Cy3 (Biotium)	13C1212
Cy5 (Biotium)	18C0626-1075
Lysine	4	LB-RS-07
2x DIGE buffer	6	LB-RS-04

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 Iso Electric Focusing

Technician:EA & NH

Solution / reagent	Batch / Lot #
1x DIGE buffer	14
Control sample	Human plasma/ SLCC1673
IPG strips	10277023
Reswell Date	22/09/2020
Time strip 1	16:10:00
Time oil added	16:40:00

Equipment	ID #
Reswell tray	3
IPGphor unit	1

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 2D SDS

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 Imaging

Technician: EA & NH

Imager: Typhoon 9400

Calibration App Log File

The imager was calibrated on 25/09/2020 following protocol P-212 and Calibration App Version 1.01.

Dye channel	PMT
Cy5	450
Cy3	455

Scanner Driver App

The gels were imaged in pairs using Scanner Driver App version 1.01. The app automatically cropped and rotated the scans. Scans were performed at 100 and 200 micron resolutions. Cy3 and Cy5 channels were imaged simultaneously.

Whole gel visual review

The gels passed a visual review (under OP-28) before upload to the cloud for processing.

Scanning App Log File

Opening excel file: A:\Lab\GelRunning\Runs\0208\Run 208.xlsx

Loaded scans:

Strip1: 49844 Strip2: 49845

Strip1: 49846 Strip2: 49847

Strip1: 49848 Strip2: 49849

Strip1: 49850 Strip2: 49851

Strip1: 49852 Strip2: 49853

Strip1: 49854 Strip2: 49855

Starting scan of Strip1: 49844 Strip2: 49845

Finished scan of Strip1: 49844 Strip2: 49845

Scanned images in 00:31:26.9429270:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49844_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49844_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49845_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49845_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49844_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49844_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49845_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49845_Cy5 560_Cy3 590 Cy5.gel

File 49844_Cy5 560_Cy3 590 Cy3.gel had 77362 saturated pixels of 5304000

File 49845_Cy5 560_Cy3 590 Cy3.gel had 107383 saturated pixels of 5304000

Starting scan of Strip1: 49846 Strip2: 49847

Finished scan of Strip1: 49846 Strip2: 49847

Scanned images in 00:31:16.6743397:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49846_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49846_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49847_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49847_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49846_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49846_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49847_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49847_Cy5 560_Cy3 590 Cy5.gel

File 49846_Cy5 560_Cy3 590 Cy3.gel had 99403 saturated pixels of 5304000

File 49847_Cy5 560_Cy3 590 Cy3.gel had 85804 saturated pixels of 5304000

Starting scan of Strip1: 49848 Strip2: 49849

Finished scan of Strip1: 49848 Strip2: 49849

Scanned images in 00:31:19.2494869:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49848_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49848_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49849_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49849_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49848_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49848_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49849_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49849_Cy5 560_Cy3 590 Cy5.gel

File 49848_Cy5 560_Cy3 590 Cy3.gel had 97777 saturated pixels of 5304000

File 49849_Cy5 560_Cy3 590 Cy3.gel had 61913 saturated pixels of 5304000

Starting scan of Strip1: 49850 Strip2: 49851

Finished scan of Strip1: 49850 Strip2: 49851

Scanned images in 00:31:20.2025415:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49850_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49850_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49851_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49851_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49850_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49850_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49851_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49851_Cy5 560_Cy3 590 Cy5.gel

File 49850_Cy5 560_Cy3 590 Cy3.gel had 97818 saturated pixels of 5304000

File 49851_Cy5 560_Cy3 590 Cy3.gel had 69973 saturated pixels of 5304000

Starting scan of Strip1: 49852 Strip2: 49853

Finished scan of Strip1: 49852 Strip2: 49853

Scanned images in 00:31:18.1004212:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49852_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49852_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49853_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49853_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49852_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49852_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49853_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49853_Cy5 560_Cy3 590 Cy5.gel

File 49852_Cy5 560_Cy3 590 Cy3.gel had 81429 saturated pixels of 5304000

File 49853_Cy5 560_Cy3 590 Cy3.gel had 82430 saturated pixels of 5304000

Starting scan of Strip1: 49854 Strip2: 49855

Finished scan of Strip1: 49854 Strip2: 49855

Scanned images in 00:31:19.8435209:

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49854_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49854_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49855_Cy5 500_Cy3 530 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\200\49855_Cy5 500_Cy3 530 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49854_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49854_Cy5 560_Cy3 590 Cy5.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49855_Cy5 560_Cy3 590 Cy3.gel

c:\Biosignatures data\2020-09-25-Run 208\9400\49855_Cy5 560_Cy3 590 Cy5.gel

File 49854_Cy5 560_Cy3 590 Cy3.gel had 89075 saturated pixels of 5304000

File 49855_Cy5 560_Cy3 590 Cy3.gel had 71489 saturated pixels of 5304000